Recovery the juvenile characteristics of mature wood by rejuvenation treatment is an important approach to improve the adventitious rooting capacity of difficulttoroot woody plants. However, the mechanism of rejuvenation is still unclear. In this study, rejuvenated and mature cuttings of walnuts were used as materials, DNA methylation pattern of both cuttings was detected at the genomewide level by methylation modificationdependent endonuclease sequencing technology (MethylRADSeq). And then, the expression level of genes which is related to differential methylation sites were analyzed. The results showed that： (1) rejuvenation treatment could significantly reduce the level of DNA methylation in walnut cuttings. (2) Functional enrichment analysis showed that genes related to differential methylation sites were mainly involved in brassinolide signal transduction, secondary metabolite biosynthesis and lignin biosynthesis. They participated in metabolic pathways such as photosynthesis, MAPK (Mitogenactivated protein kinase, MAPK) signaling pathway, fructose and mannose metabolism, cAMP (cyclic AMP, CAMP) signaling pathway and phenylpropane biosynthesis pathway. (3) qRTPCR analysis showed that key regulatory genes (NAC1, ARF5, ARF6 and WRKY22) had different expression patterns in rejuvenated and adult materials. It indicated that the rejuvenation treatment affected the expression of core regulatory genes of adventitious root formation, through reduced the genomic DNA methylation level in walnut cuttings. It may be an important regulatory pathway for rejuvenation to regulate adventitious root formation in walnut.